Preparation and use of 3-pyridyl substituted-6,6-difluoro bicyclic himbacine derivatives as par-1 receptor antagonists

ABSTRACT

The present invention relates to bicyclic himbacine derivatives of the formula 
     
       
         
         
             
             
         
       
         
         
           
             or a pharmaceutically acceptable salt thereof 
           
         
         wherein: 
         R 1  is halo; —CN; alkyl; cycloalkyl; alkoxy; phenyl, which is optionally substituted one or twice independently by alkyl, halo, or —CN; or a thiophene ring, which is optionally substituted once or twice independently by alkyl. 
       
    
     The compounds of the invention are effective inhibitors of the PAR-1 receptor. The inventive compounds may be used for the treatment or prophylaxis of disease states such as ASC, secondary prevention of myocardial infarction or stroke, or PAD.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase application under 35 U.S.C.§371 of PCT Application No. PCT/US14/051404 filed Aug. 18, 2014, whichclaims priority from U.S. Provisional Application Ser. No. 61/868,618,filed Aug. 22, 2013.

FIELD OF THE INVENTION

The present invention relates to 3′-pyridyl substituted 6,6-diflurobicyclic himbacine derivatives, which are useful as protease activatedreceptor-1 (PAR-1) antagonists and might be expected to be cannabinoid(CB₂) receptor inhibitors. PAR-1 receptors are also known in the art asthrombin receptor antagonists (TRA). The inventive compounds haveutility in treating disease states such as acute coronary syndrome (ACS)(unstable angina, non-ST-segment elevation [NSTE] myocardial infarction[MI], and ST segment-elevation myocardial infarction [STEMI]), secondaryprevention of myocardial infarction or thrombotic stroke (secondaryprevention) or peripheral artery disease (PAD), which is also know inthe art as peripheral vascular disease. The present invention alsorelates to pharmaceutical compositions comprising the inventivecompounds as well as processes for their preparation.

BACKGROUND OF THE INVENTION

Thrombin is known to have a variety of activities in different celltypes. PAR-1 receptors are known to be present in such cell types ashuman platelets, vascular smooth muscle cells, endothelial cells andfibroblasts. The art indicates that PAR-1 receptor antagonists would beexpected to be useful in the treatment of thrombotic, inflammatory,atherosclerotic and fibroproliferative disorders, as well as otherdisorders in which thrombin and its receptor play a pathological role.

Thrombin receptor antagonist peptides have been identified based onstructure-activity studies involving substitutions of amino acids onthrombin receptors. In Bernatowicz et al., J. Med. Chem., 39 (1996), p.4879-4887, tetra- and pentapeptides are disclosed as being potentthrombin receptor antagonists, for exampleN-trans-cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-NH₂ andN-trans-cinnamoyl-p-fluoroPhe-p-guanidinoPhe-Leu-Arg-Arg-NH₂. Peptidethrombin receptor antagonists are also disclosed in WO 94/03479.

Cannabinoid receptors belong to the superfamily of G-protein coupledreceptors. They are classified into the predominantly neuronal CB₁receptors and the predominantly peripheral CB₂ receptors. Thesereceptors exert their biological actions by modulating adenylate cyclaseand Ca⁺² and K⁺ currents. While the effects of CB₁ receptors areprincipally associated with the central nervous system, CB₂ receptorsare believed to have peripheral effects related to bronchialconstriction, immunomodulation and inflammation. As such, the artsuggests that a selective CB₂ receptor binding agent might be expectedto have therapeutic utility in the control of diseases associated withrheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis,diabetes, osteoporosis, renal ischemia, cerebral stroke, cerebralischemia, nephritis, inflammatory disorders of the lungs andgastrointestinal tract, and respiratory tract disorders such asreversible airway obstruction, chronic asthma and bronchitis (R. G.Pertwee, Curr. Med. Chem. 6(8), (1999), 635; M. Bensaid, MolecularPharmacology, 63 (4), (2003), 908).

Himbacine, a piperidine alkaloid of the formula

has been identified as a muscarinic receptor antagonist. The totalsynthesis of (+)-himbacine is disclosed in Chackalamannil et al., J. Am.Chem. Soc., 118 (1996), p. 9812-9813.

Substituted bi- and tricyclic thrombin receptors antagonists are knownin the art to treat thrombin receptor mediated disorders such asthrombosis, atherosclerosis, restenosis, hypertension, angina pectoris,angiogenesis related disorders, arrhythmia, a cardiovascular orcirculatory disease or condition, heart failure, ACS, myocardialinfarction, glomerulonephritis, thrombotic stroke, thromboembolyticstroke, PAD, deep vein thrombosis, venous thromboembolism, acardiovascular disease associated with hormone replacement therapy,disseminated intravascular coagulation syndrome and cerebral infarction,as well as CB₂ receptor mediated disorders. U.S. Pat. No. 6,645,987 andU.S. Pat. No. 6,894,065 disclose PAR-1 receptor antagonists of thestructure:

where R¹⁰ may be groups such as H, alkyl, haloalkyl, hydroxyl, etc. andR²² may be groups such as H, optionally substituted alkyl, hydroxyl,etc. Other known substituted thrombin receptor antagonists are disclosedin WO2001/96330, U.S. Pat. No. 6,063,847, U.S. Pat. No. 6,326,380, U.S.Pat. No. 7,037,920, U.S. Pat. No. 7,488,742, U.S. Pat. No. 7,713,999,U.S. Pat. No. 7,442,712, U.S. Pat. No. 7,488,752, U.S. Pat. Nos.7,776,889, 7,888,369, U.S. Pat. No. 8,003,803 and U.S. Pat. No.8,022,088. US 2008/0090830 and Chackalamannil et al., J. Med. Chem., 49(2006), p. 5389. A PAR-1 receptor antagonist that exhibits good thrombinreceptor antagonist activity (potency) and selectivity is vorapaxar(Merck & Co., Inc.), which has the following structure:

This compound underwent clinical trials and is disclosed in U.S. Pat.No. 7,304,048. A crystalline form of the bisulfate salt of vorapaxar isdisclosed in U.S. Pat. No. 7,235,567.

WO2011/162,562 to LG Life Sciences LTD. describes a series of [6+5]fused bicycle derivatives of the general structure:

where R₅ and R₆ are inter alia both fluoro groups, as inhibitors of thePAR-1 receptor. The compounds are taught to be useful in the treatmentand prevention of thrombus, platelet aggregation, atherosclerosis,restenosis, blood coagulation, hypertension, arrhythmia, anginapectoris, heart failure, inflammation and cancer when used alone or withother cardiovascular agents.

WO2011/28420 and WO2011/28421, both to Sanofi-Aventis, disclosecompounds that are reported to be PAR-1 receptor antagonists. Thecompounds disclosed in WO2011/28420 are pyridyl-vinyl pyrazoloquinolinesderivatives and have the following general structure:

WO2011/28421 discloses tryicyclic pyridyl-vinyl-pyrrole derivatives ofthe following general structure:

PCT/US13/027383 to Merck Sharp and Dohme, Corp. discloses bicyclichimbacine derivatives of the following general structure

where R¹⁰ and R¹¹ may both be fluoro groups. These compounds are PAR-1receptor antagonists.

Applicants discovered in accordance with the present invention that theinventive compounds act as inhibitors of PAR-1 receptor and, based upontheir structure, might also act as inhibitors of the CB₂ receptor.Therefore, the inventive compounds might be expected to be useful intreating disease states associated with the inhibition of thesereceptors.

There is a need for new compounds, formulations, treatment and therapiesto treat diseases associated with the PAR-1 and CB₂ receptors. Moreover,there is a need to develop therapeutics that exhibit improvedtherapeutic profiles; for example, desirable half-life or reducedunintended effects, such as not causing drug induced (acquired) long QTsyndrome, which potentially can be fatal, or reduced drug-druginteractions (DDIs). DDIs are potentially undesirable as they can reducethe therapeutic effectiveness of an agent or increase the incidence ofunintended effects associated with the drug. It is, therefore, an objectof this invention to provide compounds useful in the treatment,prevention or amelioration of such diseases or disorders with improvedtherapeutic profiles. These and other objectives will become evidentfrom the following description.

SUMMARY OF THE INVENTION

In its many embodiments, the present invention provides for a novelgroup of bicyclic himbacine derivatives, which are PAR-1 receptorantagonists, or metabolites, stereoisomers, salts, solvates orpolymorphs thereof, processes of preparing such compounds,pharmaceutical compositions comprising one or more such compounds,processes of preparing pharmaceutical compositions comprising one ormore such compounds and potentially methods of treatment, inhibition oramelioration of one or more disease states associated with the PAR-1receptor by administering an effective amount at least one of theinventive bicyclic himbacine derivatives to a patient in need thereof.

In one aspect, the present application discloses a compound or apharmaceutically acceptable salt, metabolite, solvate, prodrug orpolymorph of said compound, said compound or pharmaceutically acceptablesalt thereof having the general structure shown in Formula I

wherein:

-   -   R¹ is halo; —CN; alkyl; cycloalkyl; alkoxy; phenyl, which is        optionally substituted one or twice independently by alkyl,        halo, or —CN; or a thiophene ring, which is optionally        substituted once or twice independently by alkyl.

Another aspect of the present invention is pharmaceutical compositionscomprising a therapeutically effective amount of at least one compoundof Formula I or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable carrier.

Another aspect of the present invention is pharmaceutical compositionscomprising a therapeutically effective amount of at least one compoundof Formula I or a pharmaceutically acceptable salt thereof, at least oneadditional cardiovascular agent and a pharmaceutically acceptablecarrier.

Another aspect of the present invention is the possible prevention ofone or more disease state associated with inhibiting the PAR-1 receptorby administering an effective amount of a compound of Formula I or apharmaceutically acceptable salt thereof to a patient in need thereof.

Another aspect of the present invention is a method of inhibitingplatelet aggregation comprising administering to a mammal an effectiveamount of a compound of Formula I or a pharmaceutically acceptable saltthereof.

It is further contemplated that the combination of the invention couldbe provided as a kit comprising in a single package at least onecompound of Formula I or a pharmaceutically acceptable salt thereof in apharmaceutical composition, and at least one separate pharmaceuticalcomposition, such as, for example a separate pharmaceutical compositioncomprising a cardiovascular agent.

The compounds of the present invention can potentially be useful in thetreatment, amelioration or prevention of one or more conditionsassociated with inhibiting the PAR-1 receptor by administering at leastone compound of Formula I or a pharmaceutically acceptable salt thereofto a mammal in need of such treatment. Conditions that could potentiallybe treated or prevented by inhibiting the PAR-1 receptor includethrombosis, atherosclerosis, restenosis, hypertension, angina pectoris,angiogenesis related disorders, arrhythmia, a cardiovascular orcirculatory disease or condition, heart failure, ACS, myocardialinfarction, glomerulonephritis, thrombotic stroke, thromboembolyticstroke, PAD, deep vein thrombosis, venous thromboembolism, acardiovascular disease associated with hormone replacement therapy,disseminated intravascular coagulation syndrome and cerebral infarction.

Another embodiment is the possible treatment, amelioration or preventionof ACS, secondary prevention of myocardial infarction or stroke, urgentcoronary revascularization, or PAD by administering at least onecompound of Formula I or a pharmaceutically acceptable salt thereof to amammal in need of such treatment.

Another embodiment of this invention is in the possible treatment,amelioration or prevention of one or more conditions associated withcardiopulmonary bypass surgery (CPB) by administering effective amountof at least one compound of Formula I or a pharmaceutically acceptablesalt thereof to a subject of said CPB surgery. CPB surgery includescoronary artery bypass surgery (CABG), cardiac valve repair andreplacement surgery, pericardial and aortic repair surgeries. Theconditions associated with CABG include bleeding, thrombotic vascularevents (such as thrombosis or restenosis), vein graft failure, arterygraft failure, atherosclerosis, angina pectoris, myocardial ischemia,acute coronary syndrome, myocardial infarction, heart failure,arrhythmia, hypertension, transient ischemic attack, cerebral functionimpairment, thromboembolic stroke, cerebral ischemia, cerebralinfarction, thrombophlebitis, deep vein thrombosis and PAD.

Another embodiment of the present invention is the possible use of acompound of Formula I or a pharmaceutically acceptable salt thereof forthe manufacture of a medicament for the treatment, amelioration orprevention of one or more conditions associated with inhibiting thePAR-1 receptor in a patient.

DETAILED DESCRIPTION

In an embodiment, the present invention provides compounds representedby structural Formula I, or pharmaceutically acceptable salt thereof,wherein the various moieties are as described as above.

Another embodiment is the following compounds:

-   -   6′-((E)-2-((3R,3aR,4R,5S,7aS)-6,6-difluoro-7a-hydroxy-3,5-dimethyl-1-oxooctahydro        isobenzofuran-4-yl)vinyl)-[3,3′-bipyridine]-2-carbonitrile (1);    -   (3R,3aR,4R,5S,7aS)-6,6-difluoro-7a-hydroxy-3,5-dimethyl-4-((E)-2-(2′-phenyl-[3,3′-bipyridin]-6-yl)vinyl)hexahydroisobenzofuran-1(3H)-one        (2)    -   (3R,3aR,4R,5S,7aS)-6,6-difluoro-7a-hydroxy-4-((E)-2-(2′-methoxy-[3,3′-bipyridin]-6-yl)vinyl)-3,5-dimethylhexahydroisobenzofuran-1(3H)-one        (3);    -   (3R,3aR,4R,5S,7aS)-4-((E)-2-(2′-chloro-[3,3′-bipyridin]-6-yl)vinyl)-6,6-difluoro-7a-hydroxy-3,5-dimethylhexahydroisobenzofuran-1(3H)-one        (4);    -   (3R,3aR,4R,5S,7aS)-4-((E)-2-(2′-fluoro-[3,3′-bipyridin]-6-yl)vinyl)-7a-hydroxy-3,5-dimethylhexahydroisobenzofuran-1(3H)-one        (5);    -   (3R,3aR,4R,5S,7aS)-4-((E)-2-(2′-cyclopropyl-[3,3′-bipyridin]-6-yl)vinyl)-6,6-difluoro-7a-hydroxy-3,5-dimethylhexahydroisobenzofuran-1(3H)-one        (6);    -   3R,3        aR,4R,5S,7aS)-6,6-difluoro-7a-hydroxy-3,5-dimethyl-4-((E)-2-(2′-(5-methylthiophen-2-yl)-[3,3′-bipyridin]-6-yl)vinyl)hexahydroisobenzofuran-1(3H)-one        (7);

or a pharmaceutically acceptable salt thereof.

Another embodiment of the present invention is the following compoundsor a pharmaceutically acceptable salt thereof wherein R¹ is —CN or halo(e.g., —F or —Cl).

Another embodiment of the present invention is the following compoundsor a pharmaceutically acceptable salt thereof wherein R¹ is cycloalkyl(e.g, cyclopropyl) or alkoxy (e.g., methoxy or ethoxy).

Another embodiment of the present invention is when R¹ is phenyl.

Another embodiment of the present invention is when R¹ is

As used above, and throughout this disclosure, the following terms,unless otherwise indicated, shall be understood to have the followingmeanings:

“Patient” or “subject” includes both humans and animals.

“Mammal” means humans and other mammalian animals.

“Alkyl” means an aliphatic hydrocarbon group which may be straight orbranched and comprising about 1 to about 20 carbon or about 1 to 12atoms in the chain. “Lower alkyl” means a group having about 1 to about6 carbon atoms in the chain which may be straight or branched. Branchedmeans that one or more lower alkyl groups such as methyl, ethyl orpropyl, are attached to a linear alkyl chain.

“Alkoxy” means an alkyl-O— group in which the alkyl group is aspreviously described. Non-limiting examples of suitable alkoxy groupsinclude methoxy, ethoxy, n-propoxy, isopropoxy and n-butoxy. The bond tothe parent moiety is through the ether oxygen.

“Halo” refers to fluorine, chlorine, bromine or iodine radicals.Preferred are fluoro, chloro or bromo, and more preferred are fluoro andchloro.

“Halogen” means fluorine, chlorine, bromine, or iodine. Non-limitingexamples include fluorine or chlorine.

“Haloalkyl” means a halo-alkyl-group in which the alkyl group is aspreviously described. The bond to the parent moiety is through thealkyl. Non-limiting examples of suitable haloalkyl groups includefluoromethyl, difluoromethyl, —CH₂CF₃, —CH₂CHF₂ or —CH₂CH₂F.

“Cycloalkyl” is a cyclized alkyl ring having 3-12 or 3-6 carbon atoms.Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl andcyclohexyl.

The term “isolated” or “in isolated form” for a compound refers to thephysical state of said compound after being isolated from a syntheticprocess or natural source or combination thereof. The term “purified” or“in purified form” for a compound refers to the physical state of saidcompound after being obtained from a purification process or processesdescribed herein or well known to the skilled artisan, in sufficientpurity to be characterizable by standard analytical techniques describedherein or well known to the skilled artisan.

When a functional group in a compound is termed “protected”, this meansthat the group is in modified form to preclude undesired side reactionsat the protected site when the compound is subjected to a reaction.Suitable protecting groups will be recognized by those with ordinaryskill in the art as well as by reference to standard textbooks such as,for example, T. W. Greene et al, Protective Groups in Organic Synthesis(1991), Wiley, N.Y.

“Effective amount” or “therapeutically effective amount” is meant todescribe an amount of compound or a composition of the present inventioneffective as PAR-1 or thrombin receptor antagonists, thereby producingthe desired therapeutic, ameliorative, inhibitory or preventativeeffect.

The compounds of Formula I can form salts which are also within thescope of this invention. Reference to a compound of Formula I herein isunderstood to include reference to salts thereof, unless otherwiseindicated. The term “salt(s)”, as employed herein, denotes acidic saltsformed with inorganic and/or organic acids, as well as basic saltsformed with inorganic and/or organic bases. In addition, when a compoundof Formula I contains both a basic moiety, such as, but not limited to apyridine or imidazole, and an acidic moiety, such as, but not limited toa carboxylic acid, zwitterions (“inner salts”) may be formed and areincluded within the term “salt(s)” as used herein. Pharmaceuticallyacceptable (i.e., non-toxic, physiologically acceptable) salts arepreferred, although other salts are also useful. Salts of the compoundsof the Formula I may be formed, for example, by reacting a compound ofFormula I with an amount of acid or base, such as an equivalent amount,in a medium such as one in which the salt precipitates or in an aqueousmedium followed by lyophilization.

Exemplary acid addition salts include acetates, ascorbates, benzoates,benzenesulfonates, bisulfates, borates, butyrates, citrates,camphorates, camphorsulfonates, fumarates, hydrochlorides,hydrobromides, hydroiodides, lactates, maleates, methanesulfonates,naphthalenesulfonates, nitrates, oxalates, phosphates, propionates,salicylates, succinates, sulfates, tartarates, thiocyanates,toluenesulfonates (also known as tosylates,) and the like. Additionally,acids which are generally considered suitable for the formation ofpharmaceutically useful salts from basic pharmaceutical compounds arediscussed, for example, by P. Stahl et al, Camille G. (eds.) Handbook ofPharmaceutical Salts. Properties, Selection and Use. (2002) Zurich:Wiley-VCH; S. Berge et al, Journal of Pharmaceutical Sciences (1977)66(1) 1-19; P. Gould, International J. of Pharmaceutics (1986) 33201-217; Anderson et al, The Practice of Medicinal Chemistry (1996),Academic Press, New York; and in The Orange Book (Food & DrugAdministration, Washington, D.C. on their website). These disclosuresare incorporated herein by reference thereto.

Exemplary basic salts include ammonium salts, alkali metal salts such assodium, lithium, and potassium salts, alkaline earth metal salts such ascalcium and magnesium salts, salts with organic bases (for example,organic amines) such as dicyclohexylamines, t-butyl amines, and saltswith amino acids such as arginine, lysine and the like. Basicnitrogen-containing groups may be quarternized with agents such as loweralkyl halides (e.g. methyl, ethyl, and butyl chlorides, bromides andiodides), dialkyl sulfates (e.g. dimethyl, diethyl, and dibutylsulfates), long chain halides (e.g. decyl, lauryl, and stearylchlorides, bromides and iodides), aralkyl halides (e.g. benzyl andphenethyl bromides), and others.

All such acid salts and base salts are intended to be pharmaceuticallyacceptable salts within the scope of the invention and all acid and basesalts are considered equivalent to the free forms of the correspondingcompounds for purposes of the invention.

In this application, unless otherwise indicated, whenever there is astructural formula provided, such as those of Formula I, this formula isintended to encompass all forms of a compound such as, for example, anysolvates, hydrates, stereoisomers, tautomers, co-crystals, polymorphsetc.

Compounds of Formula I, and salts, solvates, co-crystals and prodrugsthereof, may exist in their tautomeric form (for example, as an amide orimino ether). All such tautomeric forms are contemplated herein as partof the present invention.

Prodrugs, solvates and co-crystals of the compounds of the invention arealso contemplated herein. The term “prodrug”, as employed herein,denotes a compound that is a drug precursor which, upon administrationto a subject, undergoes chemical conversion by metabolic or chemicalprocesses to yield a compound of Formula I or a salt thereof. Adiscussion of prodrugs is provided in T. Higuchi and V. Stella,Pro-drugs as Novel Delivery Systems (1987) 14 of the A.C.S. SymposiumSeries, and in Bioreversible Carriers in Drug Design, (1987) Edward B.Roche, ed., American Pharmaceutical Association and Pergamon Press, bothof which are incorporated herein by reference thereto.

“Solvate” means a physical association of a compound of this inventionwith one or more solvent molecules. This physical association involvesvarying degrees of ionic and covalent bonding, including hydrogenbonding. In certain instances the solvate will be capable of isolation,for example when one or more solvent molecules are incorporated in thecrystal lattice of the crystalline solid. “Solvate” encompasses bothsolution-phase and isolatable solvates. Non-limiting examples ofsuitable solvates include ethanolates, methanolates, and the like.“Hydrate” is a solvate wherein the solvent molecule is H₂O.

A co-crystal is a crystalline superstructure formed by combining anactive pharmaceutical intermediate with an inert molecule and providescrystallinity to the combined form. Co-crystals are often made between adicarboxlyic acid such as fumaric acid, succinic acid etc. and a basicamine, such as the one represented by a compound of this invention indifferent proportions depending on the nature of the co-crystal.(Remenar, J. F. et. al. J Am. Chem. Soc. 2003, 125, 8456).

All stereoisomers (for example, geometric isomers, optical isomers andthe like) of the present compounds (including those of the salts,solvates, co-crystals and prodrugs of the compounds as well as the saltsand solvates, co-crystals of the prodrugs), such as those which mayexist due to asymmetric carbons on various substituents, includingenantiomeric forms (which may exist even in the absence of asymmetriccarbons), rotameric forms, atropisomers, and diastereomeric forms, arecontemplated within the scope of this invention, as are positionalisomers (such as, for example, 4-pyridyl and 3-pyridyl). Individualstereoisomers of the compounds of the invention may, for example, besubstantially free of other isomers, or may be admixed, for example, asracemates or with all other, or other selected, stereoisomers. Thechiral centers of the present invention can have the S or Rconfiguration as defined by the IUPAC 1974 Recommendations. The use ofthe terms “salt”, “solvate” “prodrug” and the like, is intended toequally apply to the salt, solvate and prodrug of enantiomers,stereoisomers, rotamers, tautomers, positional isomers, racemates orprodrugs of the inventive compounds.

The present invention also embraces isotopically-labelled compounds ofthe present invention which are identical to those recited herein, butfor the fact that one or more atoms are replaced by an atom having anatomic mass or mass number different from the atomic mass or mass numberusually found in nature. Examples of isotopes that can be incorporatedinto compounds of the invention include isotopes of hydrogen, carbon,nitrogen, oxygen, phosphorus, fluorine and chlorine and iodine, such as²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F, ³⁶Cl and ¹²³I,respectively.

Certain isotopically-labelled compounds of Formula (I) (e.g., thoselabeled with ³H and ¹⁴C) are useful in compound and/or substrate tissuedistribution assays. Tritiated (i.e., ³H) and carbon-14 (i.e., ¹⁴C)isotopes are particularly preferred for their ease of preparation anddetectability. Certain isotopically-labelled compounds of Formula (I)can be useful for medical imaging purposes. E.g., those labeled withpositron-emitting isotopes like ¹¹C or ¹⁸F can be useful for applicationin Positron Emission Tomography (PET) and those labeled with gamma rayemitting isotopes like ¹²³I can be useful for application in Singlephoton emission computed tomography (SPECT). Further, substitution withheavier isotopes such as deuterium (i.e., ²H) may afford certaintherapeutic advantages resulting from greater metabolic stability (e.g.,increased in vivo half-life or reduced dosage requirements) and hencemay be preferred in some circumstances. Additionally, isotopicsubstitution at a site where epimerization occurs may slow or reduce theepimerization process and thereby retain the more active or efficaciousform of the compound for a longer period of time. Isotopically-labeledcompounds of Formula (I), in particular those containing isotopes withlonger half lives (T½>1 day), can generally be prepared by followingprocedures analogous to those disclosed in the Schemes and/or in theExamples herein below, by substituting an appropriate isotopicallylabeled reagent for a non-isotopically labeled reagent.

As discussed above, the compounds of Formula I may be used to treat,ameliorate or prevent conditions associated with inhibiting the PAR-1receptor. In addition to the conditions mentioned above, otherconditions could include migraine, erectile dysfunction, rheumatoidarthritis, rheumatism, astrogliosis, a fibrotic disorder of the liver,kidney, lung or intestinal tract, systemic lupus erythematosus, multiplesclerosis, osteoporosis, renal disease, acute renal failure, chronicrenal failure, renal vascular homeostasis, renal ischemia, bladderinflammation, diabetes, diabetic neuropathy, cerebral stroke, cerebralischemia, nephritis, cancer, melanoma, renal cell carcinoma, neuropathy,malignant tumors, neurodegenerative and/or neurotoxic diseases,conditions or injuries, Alzheimer's disease, an inflammatory disease orcondition, asthma, glaucoma, macular degeneration, psoriasis,endothelial dysfunction disorders of the liver, kidney or lung,inflammatory disorders of the lungs and gastrointestinal tract,respiratory tract disease or condition, radiation fibrosis, endothelialdysfunction, periodontal diseases or wounds, or a spinal cord injury, ora symptom or result thereof, viral infections, including infections fromhuman respiratory syncytial virus (hRSV), human metapneumovirus (hMPV)and influenza virus type A, as well as other disorders in which thrombinand its receptor play a pathological role.

In addition to their PAR-1 receptor antagonist properties, the compoundsof Formula I or the pharmaceutically acceptable salts might be expectedto be used to treat, ameliorate or prevent one or more conditionsassociated with inhibiting the CB₂ receptor by administering at leastone compound of Formula I or a pharmaceutically acceptable salt thereofto a mammal in need of such treatment. Conditions might include, forexample, rheumatoid arthritis, systemic lupus erythematosus, multiplesclerosis, diabetes, osteoporosis, renal ischemia, cerebral stroke,cerebral ischemia, nephritis, inflammatory disorders of the lungs andgastrointestinal tract, and respiratory tract disorders such asreversible airway obstruction, chronic asthma and bronchitis.

In another embodiment, compounds of the present invention might beexpected to be useful in a method for treating, ameliorating orpreventing radiation- and/or chemical-induced toxicity in non-malignanttissue in a patient comprising administering a therapeutically effectiveamount of at least one compound of Formula I or a pharmaceuticallyacceptable salt thereof. In particular, the radiation- and/orchemical-induced toxicity is one or more of intestinal fibrosis,pneumonitis, and mucositis. In one embodiment, the radiation- and/orchemical-induced toxicity is intestinal fibrosis. In another embodiment,the radiation- and/or chemical-induced toxicity is oral mucositis. Inyet another embodiment, the radiation- and/or chemical-induced toxicityis intestinal mucositis, intestinal fibrosis, intestinal radiationsyndrome, or pathophysiological manifestations of intestinal radiationexposure.

The present invention might also be expected to provides for methods forreducing structural radiation injury in a patient that will be exposed,is concurrently exposed, or was exposed to radiation and/or chemicaltoxicity, comprising administering a therapeutically effective amount ofat least one compound of Formula I or a pharmaceutically acceptable saltthereof. The present invention might also be expected to provide formethods for reducing inflammation in a patient that will be exposed, isconcurrently exposed, or was exposed to radiation and/or chemicaltoxicity, comprising administering a therapeutically effective amount ofat least one compound of Formula I or a pharmaceutically acceptable saltthereof. The present invention might also be expected to provide formethods for adverse tissue remodeling in a patient that will be exposed,is concurrently exposed, or was exposed to radiation and/or chemicaltoxicity, comprising administering a therapeutically effective amount ofat least one compound of Formula I or a pharmaceutically acceptable saltthereof. The present invention might also be expected to provide formethods for reducing fibroproliferative tissue effects in a patient thatwill be exposed, is concurrently exposed, or was exposed to radiationand/or chemical toxicity, comprising administering a therapeuticallyeffective amount of at least one compound of Formula I or apharmaceutically acceptable salt thereof.

The present invention might also be expected to provide for methodsuseful for treating a cell proliferative disorder in a patient sufferingtherefrom comprising administering a therapeutically effective amount ofat least one compound of Formula I or a pharmaceutically acceptable saltthereof. In one embodiment, the cell proliferative disorder ispancreatic cancer, glioma, ovarian cancer, colorectal and/or coloncancer, breast cancer, prostate cancer, thyroid cancer, lung cancer,melanoma, or stomach cancer. In one embodiment, the glioma is ananaplastic astrocytoma. In another embodiment, the glioma is aglioblastoma multiforme.

As used above, the term inflammatory disease or condition includesirritable bowel syndrome, Crohn's disease, nephritis or a radiation- orchemotherapy-induced proliferative or inflammatory disorder of thegastrointestinal tract, lung, urinary bladder, gastrointestinal tract orother organ. The term respiratory tract disease or condition includesreversible airway obstruction, asthma, chronic asthma, bronchitis orchronic airways disease. “Cancer” includes renal cell carcinoma or anangiogenesis related disorder. “Neurodegenerative disease” includesParkinson's disease, amyotropic lateral sclerosis, Alzheimer's disease,Huntington's disease or Wilson's disease.

The term “pharmaceutical composition” is also intended to encompass boththe bulk composition and individual dosage units comprised of more thanone (e.g., two) pharmaceutically active agents such as, for example, acompound of the present invention and an additional agent selected fromthe lists of the additional agents described herein, along with anypharmaceutically acceptable carrier. The bulk composition and eachindividual dosage unit can contain fixed amounts of the afore-said “morethan one pharmaceutically active agents”. The bulk composition ismaterial that has not yet been formed into individual dosage units. Anillustrative dosage unit is an oral dosage unit such as tablets, pillsand the like. Similarly, the herein-described method of treating apatient by administering a pharmaceutical composition of the presentinvention is also intended to encompass the administration of theafore-said bulk composition and individual dosage units.

The amount and frequency of administration of the compound of thisinvention and/or their pharmaceutically acceptable salts will beregulated according to the judgment of the attending clinicianconsidering such factors as age, condition and size of the patient aswell as the severity of the symptoms being treated.

The quantity of active compound in a unit dose of preparation may bevaried or adjusted from about 1 mg to about 150 mg, preferably fromabout 1 mg to about 75 mg, more preferably from about 1 mg to about 50mg, according to the particular application.

The term “patient” includes animals, preferably mammals and especiallyhumans, who use the instant active agents for the prevention ortreatment of a medical condition. Administering of the drug to thepatient includes both self-administration and administration to thepatient by another person. The patient may be in need of treatment foran existing disease or medical condition, or may desire prophylactictreatment to prevent or reduce the risk of said disease or medicalcondition.

The term “therapeutically effective amount” is intended to mean thatamount of a drug or pharmaceutical agent that will elicit the biologicalor medical response of a tissue, a system, animal or human that is beingsought by a researcher, veterinarian, medical doctor or other clinician.A “prophylactically effective amount” is intended to mean that amount ofa pharmaceutical drug that will prevent or reduce the risk of occurrenceof the biological or medical event that is sought to be prevented in atissue, a system, animal or human by a researcher, veterinarian, medicaldoctor or other clinician. The terms “preventing” or “prevention” areused herein to refer to administering a compound before the onset ofclinical symptoms.

The compounds of this invention may also be useful in combination(administered together or sequentially) with one or more therapeuticagents, such as, for example, another cardiovascular agent.Cardiovascular agents that could be used in combination with thecompounds for Formula I or their pharmaceutically acceptable saltsinclude drugs that have anti-thrombotic, anti-platelet aggregation,antiatherosclerotic, antirestenotic and/or anti-coagulant activity. Suchdrugs are useful in treating thrombosis-related diseases includingthrombosis, atherosclerosis, restenosis, hypertension, angina pectoris,arrhythmia, heart failure, myocardial infarction, glomerulonephritis,thrombotic and thromboembolic stroke, peripheral vascular diseases,other cardiovascular diseases, cerebral ischemia, inflammatory disordersand cancer, as well as other disorders in which thrombin and itsreceptor play a pathological role. Suitable cardiovascular agents areselected from the group consisting of thromboxane A2 biosynthesisinhibitors such as aspirin; thromboxane antagonists such as seratrodast,picotamide and ramatroban; adenosine diphosphate (ADP) inhibitors suchas clopidogrel; cyclooxygenase inhibitors such as aspirin, meloxicam,rofecoxib and celecoxib; angiotensin antagonists such as valsartan,telmisartan, candesartran, irbesartran, losartan and eprosartan;endothelin antagonists such as tezosentan; phosphodiesterase inhibitorssuch as milrinoone and enoximone; angiotensin converting enzyme (ACE)inhibitors such as captopril, enalapril, enaliprilat, spirapril,quinapril, perindopril, ramipril, fosinopril, trandolapril, lisinopril,moexipril and benazapril; neutral endopeptidase inhibitors such ascandoxatril and ecadotril; anticoagulants such as ximelagatran,fondaparin and enoxaparin; diuretics such as chlorothiazide,hydrochlorothiazide, ethacrynic acid, furosemide and amiloride; plateletaggregation inhibitors such as abciximab and eptifibatide; and GPIIb/IIIa antagonists.

Other possible combinations might include lipid lowering agents (e.g.,simvastatin, lovastatin, pravastatin, atorvastatin rosuvastatin,pitavastatin, ezetimibe); niacin in immediate-release or controlledrelease forms or niacin in combination with a DP antagonist, such aslaropiprant and/or with an HMG-CoA reductase inhibitor; niacin receptoragonists such as acipimox and acifran, as well as niacin receptorpartial agonists; metabolic altering agents including insulinsensitizing agents and related compounds (e.g., muraglitazar, glipizide,stigliptin, metformin, rosiglitazone statins, e.g., simvastatin,atorvastatin and rosovastatin), PCSK9 inhibitors, e.g.antibodies—REGN727, AMG-145, RN316, RG7652; and small moleculeinhibitors and CETP inhibitors, e.g., anacetrapib, evacetrapib, etc.Other possible combinations include AMPK agonists (e.g., ETC-1002);glucagon receptor antagonists; Lp-PLA2 inhibitors (e.g., darapladib) andanti-IL-1beta antibodies (canakinumab).

The dosage of the cardiovascular agent can be determined from publishedmaterial, and may range from 1 to 1000 mg per dose.

An embodiment of this invention is combinations comprising an effectiveamount of a compound of Formula I or a pharmaceutically acceptable saltthereof and an ADP antagonist and/or cyclooxygenase inhibitor.

Non-limiting combinations comprise an effective amount of a compoundaccording to Formula I or a pharmaceutically acceptable salt thereof andaspirin, ticagrelor, cangrelor, clopidogrel (either as a free base or asa pharmaceutically acceptable salt, such as its bisulfate salt),prasugrel, ticlopidine or fragmin.

Other therapeutic agents could include drugs that are known and used inthe treatment of inflammation, rheumatism, asthma, glomerulonephritis,osteoporosis, neuropathy and/or malignant tumors, angiogenesis relateddisorders, cancer, disorders of the liver, kidney and lung, melanoma,renal cell carcinoma, renal disease, acute renal failure, chronic renalfailure, renal vascular homeostasis, glomerulonephritis, chronic airwaysdisease, bladder inflammation, neurodegenerative and/or neurotoxicdiseases, conditions, or injuries, radiation fibrosis, endothelialdysfunction, periodontal diseases and wounds. Further examples oftherapeutically effective agents which may be administered incombination with a compound of Formula I or a pharmaceuticallyacceptable salt thereof include resistance factors for tumor cellstowards chemotherapy and proliferation inhibitors of smooth musclecells, endothelial cells, fibroblasts, kidney cells, osteosarcoma cells,muscle cells, cancer cells and/or glial cells.

For treating and/or preventing radiation- and/or chemical-inducedtoxicity in non-malignant tissue, the present invention includesadministering to a patient in need of such treatment an effective amountof a combination of one or more compounds of formula I and one or moreradiation-response modifiers selected from the group consisting ofKepivance™ (palifermin), L-glutamine, teduglutide, sucralfate mouthrinses, iseganan, lactoferrin, mesna and trefoil factor.

For treating a cell proliferative disorder the present inventionincludes administering to a patient in need of such tretment aneffective amount of a combination of one or more compounds of Formula Ior a pharmaceutically acceptable salt thereof and another antineoplasticagent. In one embodiment, the other antineoplastic agent is temozolomideand the cell proliferative disorder is glioma. In another embodiment,the other antineoplastic agent is interferon and the cell proliferativedisorder is melanoma. In one embodiment, the other antineoplastic agentis PEG-Intron (peginterferon alpha-2b) and the cell proliferativedisorder is melanoma.

Pharmaceutical compositions comprising a therapeutically effectiveamount of a combination of at least one compound of Formula I or apharmaceutically acceptable salt thereof and a radiation-responsemodifier in a pharmaceutically acceptable carrier are also provided.

Pharmaceutical compositions comprising a therapeutically effectiveamount of a combination of at least one compound of Formula I or apharmaceutically acceptable salt thereof and an antineoplastic agent ina pharmaceutically acceptable carrier are also provided.

For preparing pharmaceutical compositions from the compounds describedby this invention, inert, pharmaceutically acceptable carriers can beeither solid or liquid. Solid form preparations include powders,tablets, dispersible granules, capsules, cachets and suppositories. Thepowders and tablets may be comprised of from about 5 to about 95 percentactive ingredient. Suitable solid carriers are known in the art, e.g.magnesium carbonate, magnesium stearate, talc, sugar or lactose.Tablets, powders, cachets and capsules can be used as solid dosage formssuitable for oral administration. Examples of pharmaceuticallyacceptable carriers and methods of manufacture for various compositionsmay be found in A. Gennaro (ed.), The Science and Practice of Pharmacy,20^(th) Edition, (2000), Lippincott Williams & Wilkins, Baltimore, Md.

Liquid form preparations include solutions, suspensions and emulsions.As an example may be mentioned water or water-propylene glycol solutionsfor parenteral injection or addition of sweeteners and opacifiers fororal solutions, suspensions and emulsions. Liquid form preparations mayalso include solutions for intranasal administration.

Aerosol preparations suitable for inhalation may include solutions andsolids in powder form, which may be in combination with apharmaceutically acceptable carrier, such as an inert compressed gas,e.g. nitrogen.

Also included are solid form preparations which are intended to beconverted, shortly before use, to liquid form preparations for eitheroral or parenteral administration. Such liquid forms include solutions,suspensions and emulsions.

The compounds of the invention may also be deliverable transdermally.The transdermal compositions can take the form of creams, lotions,aerosols and/or emulsions and can be included in a transdermal patch ofthe matrix or reservoir type as are conventional in the art for thispurpose.

Preferably the compound is administered orally.

Preferably, the pharmaceutical preparation is in a unit dosage form. Insuch form, the preparation is subdivided into suitably sized unit dosescontaining appropriate quantities of the active component, e.g., aneffective amount to achieve the desired purpose.

In general, the compounds in the invention may be produced by a varietyof processes know to those skilled in the art and by know processesanalogous thereto. The invention disclosed herein is exemplified by thefollowing preparations and examples which should not be construed tolimit the scope of the disclosure. Alternative mechanistic pathways andanalogous structures will be apparent to those skilled in the art. Thepractitioner is not limited to these methods.

Moreover, one skilled in the art would have resources such as ChemicalAbstracts or Beilstein at his or her disposal to assist in preparing aspecific compound.

One skilled in the art will recognize that one route will be optimizeddepending on the choice of appendage substituents. Additionally, oneskilled in the art will recognize that in some cases the order of stepshas to be controlled to avoid functional group incompatibility.

The prepared compounds may be analyzed for their composition and purityas well as characterized by standard analytical techniques such as, forexample, elemental analysis, NMR, mass spectroscopy and IR spectra.

One skilled in the art will recognize that reagents and solventsactually used may be selected from several reagents and solvents wellknown in the art to be effective equivalents. Hence, when a specificsolvent or reagent is mentioned, it is meant to be an illustrativeexample of the conditions desirable for that particular reaction schemeand in the preparations and examples described below.

Where NMR data are presented, 1H spectra were obtained, for example, oneither a Varian Inova (400 or 500 mHz), Varian Mercury VX-400 (400 MHz),or Bruker-Biospin AV-500 (500 MHz), and are reported as ppm with numberof protons and multiplicities indicated parenthetically. Where LC/MSdata are presented, analyses was performed, for example, using anAgilent 1100 series or Applied Biosystems® API-100 mass spectrometer andC18 column, 5-95% CH₃CN—H₂O (with 0.05% TFA) gradient. The observedparent ion is given

Throughout the synthetic schemes, abbreviations are used with thefollowing meaning unless otherwise indicated:

-   ACN or MeCN=acetonitrile; Ac₂O=acetic anhydride; Aq.=aqueous;    t-Butyl=tert-butyl; t-BuOH=tert-butyl alcohol; cat.=catalyst;    DBU=1,8-Diazabicyclo[5.4.0]undec-7-ene;    DCC=N,N′-bicyclohexylcarbodiimide; DCM=dichloromethane;    DAST=diethylaminosulfur trifluoride; DMAC=N,N-dimethylacetamide;    DMAP=4-dimethylamino pyridine; DMEM=Dulbecco's modified eagle mediu,    DMF=dimethylformamide; DMP=Dess-Martin periodinane;    DMSO=dimethylsulfoxide; DIEA=N,N-Diisopropylethylamine or Hünig's    base; Et=ethyl; EtOH=ethanol; EtOAc=ethyl acetate; g=gas    HATU=O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate;    HEPES=(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid);    HPLC=high pressure liquid chromatography; HOAc=acetic acid;    LCMS=liquid chromatorgraphy-mass spectrometry; KHMDS=Potassium    bis(trimethylsilyl)amide; LiHMDS=lithium bis(trimethylsilyl)amide;    Me=methyl; MeOH=methanol; Mel=methyl iodide; mmol=millimoles    MPLC=medium pressure liquid chromatography; Ms=mesylate; MS    ESI=electrospray ionixation mass spectrometry; MTBE=methyl    tert-butyl ether; NMM=N-methylmorpholine;    NMP=N-methyl-2-pyrrolidone; Ph=phenyl; piv-cl=pivaloyl chloride;    i-Pr=iso-propyl; RT or rt=room temperature TEA=triethanolamine;    TFA=trifluoroacetic acid; THF=tetrahydrofuran; TLC=thin layer    chromatography.

INTERMEDIATE SYNTHESES

Intermediate compounds of the present invention can be synthesizedaccording to the schemes and procedures outlined below. Since theschemes are an illustration, the invention should not be construed asbeing limited by the chemical reactions and conditions expressed. Thepreparation of the various starting materials used in the schemes iswell within the ordinary skill level of a practitioner of this art.Unless otherwise indicated, the definition for a variable is the same asthat provided in Formula I.

Intermediate A can be prepared from commerically available and knownstarting materials according to Scheme A. 2-Ethylfuran (A-1) wasoxidized to the corresponding hydroxyfuranone, which under the action ofbase opened to carboxylic acid (A-2). Alkynylalcohol (A-3) was reducedto the corresponding cis-alkene (A-4) using Lindlar's catalyst under anatmosphere of hydrogen gas. DCC-mediated coupling of preparedintermediates A-2 and A-4 provides the complete carbon framework forintermediate A in compound (A-5). Formation of the enol acetate (A-6)sets the stage for an intramolecular Diels-Alder reaction to formlactone (A-7). Hydrolysis and subsequent reaction of ketone (A-8) withDAST provided the C6-difluoro lactone (A-9). Saponification of the amideprovided carboxylic acid (A-10), which was then chemoselectively reducedvia a two-step protocol to yield alcohol (A-11). Oxidation of alcohol(A-11) to aldehyde (A-12) and a Horner-Wadsworth-Emmons olefinationreaction with known phosphonate ester (A-13) provided Intermediate A.

Intermediate A

Step 1: 5-ethyl-5-hydroxyfuran-2(5H)-one: NaH₂PO₄ (243 g, 3.12 mol) wasadded to a solution of 2-ethylfuran (100 g, 1.04 mol) in t-BuOH (1.0 L)and H₂O (200 mL) at room temperature. After 30 min, NaClO₂ (312 g, 3.12mol) was added portionwise. The temperature was controlled between10-30° C. After the addition, the reaction was stirred for another 2 huntil the reaction went to completion. The reaction solution was purgedwith N₂ overnight until it turned to white. The white precipitate wasfiltered and t-BuOH was removed under vacuo. The reaction was extractedwith CH₂C1₂ and dried with anhydrous Na₂SO₄, providing the titlecompound, which was used directly for the next step without furtherpurification.

Step 2: (E)-4-oxohex-2-enoic acid: To a solution of5-ethyl-5-hydroxyfuran-2(5H)-one (130 g, 1.02 mol) in THF (645 mL) wasadded acetone (520 mL), water (130 mL), pyridine (8.1 mL, 0.1 mol) atroom temperature. The reaction was stirred overnight. TLC (petroleumether/ethyl acetate, 3:1) showed the reaction was completed. The mixturewas concentrated under vacuo. The residue was treated with 10% K₂CO₃ topH>10 at 0° C. and extracted with ethyl acetate (500 mL×3). The aqueouslayer was acidified with concentrated HCl at 0° C. to pH<2. Afterextraction with ethyl acetate (500 mL×6) and washed with brine, theorganic layer was dried with anhydrous Na₂SO₄ and concentrated to givecrude product. A final wash with methyl tert-butyl ether provided thetitle compound. ¹H NMR (400 MHz, CDCl₃) δ 7.12 (d, 1H, J=16.0 Hz), 6.66(d, 1H, J=15.6 Hz), 2.68 (q, 2H, J=7.2 Hz), 1.13 (q, 2H, J=7.2 Hz).

Step 3: (R, Z)-4-hydroxy-N,N-diphenylpent-2-enamide: To a solution of(R)-4-hydroxy-N,N-diphenylpent-2-ynamide (200 g, 0.75 mol) and Lindlarcatalyst (13.6 g, 7.5 mmol) in CH₃OH (2 L) was added quinoline (21.6 mL,182 mmol) at room temperature. The reaction was evacuated and rechargedwith a balloon of H₂. After stirring at room temperature for 1 h, TLC(petroleum ether/ethyl acetate, 3:1) showed the reaction was complete.Solvent was removed under reduced pressure at 35° C. THF (1 L) was addedwhich was followed by the addition of petroleum ether (1 L). Afterremoving half amount of the solvent, petroleum ether (1 L) was added. Aprecipicate formed during concentration, which was filtered and washedwith methyl tert-butyl ether to afford the title compound. The combinedfiltrate residues were purified by silica gel column chromatography(petroleum ether:ethyl acetate, 5:1) to yield another batch of the titlecompound. ¹H NMR (400 MHz, CDCl₃) δ 7.38-7.23 (m, 10H), 6.09 (dd, 1H,J=12.0, 6.0 Hz), 5.82 (d, 1H, J=12.0 Hz), 4.88-4.85 (m, 1H), 1.35 (d,1H, J=6.8 Hz).

Step 4: (E)-(R,Z)-5-(diphenylamino)-5-oxopent-3-en-2-yl4-oxohex-2-enoate: NMM (91 mL, 814 mmol) was added to(E)-4-oxohex-2-enoic acid (58 g, 450 mmol) in anhydrous toluene (800 mL)at 0° C. Then, pivaloyl chloride (55 mL, 450 mmol) was added dropwisewhile maintaining the internal temperature between 0-5° C. After theaddition, the reaction was stirred at 0° C. for 30 min. (R,Z)-4-Hydroxy-N,N-diphenylpent-2-enamide (100 g, 370 mmol) and DMAP (4.57g, 37 mmol) in anhydrous toluene (400 mL) and anhydrous THF (200 mL)were added dropwise to the reaction mixture while maintaining thetemperature between 0-5° C. under N₂. After 2 hours, the TLC (petroleumether:ethyl acetate, 5:1) showed that the reaction was complete. 9 NH₂SO₄ (330 mL) was added dropwise to quench the reaction, while thetemperature was kept between 0-5° C. The reactions were combinedtogether and extracted with methyl tert-butyl ether and washed withsaturated NaHCO₃. The crude product was purified by silica gel columnchromatography (petroleum ether:ethyl acetate, 15:1) to give the titlecompound. ¹H NMR (400 MHz, CDCl₃) δ 7.34-7.24 (m, 10H), 7.06 (dd, 1H,J=16.0, 4.0 Hz), 6.66 (dd, 1H, J=16.0, 4.0 Hz), 6.35-6.32 (m, 1H),5.88-5.80 (m, 2H), 2.68-2.63 (m, 2H), 1.49-1.47 (m, 3H), 1.13-1.08 (m,3H).

Step 5: (2E,4Z)-(R,Z)-5-(diphenylamino)-5-oxopent-3-en-2-yl4-acetoxyhexa-2,4-dienoate:(E)-(R,Z)-5-(Diphenylamino)-5-oxopent-3-en-2-yl 4-oxohex-2-enoate (100g, 265 mmol) and DMAP (9.5 g, 79 mmol) in Ac₂O (100 mL, 1.06 mol) werestirred at 50° C. for 19 h. TLC (petroleum ether:ethyl acetate, 5:1)showed the reaction was complete. The reaction was concentrated underreduced pressure at 45° C. The reaction was extracted with methyltert-butyl ether and washed with 10% citric acid (5 L). The organiclayer was washed with sat. NaHCO₃, brine, dried over Na₂SO₄ andfiltered, the reaction was concentrated to give the title compound,which was used without further purification in the next step. ¹H NMR(400 MHz, CDCl₃) δ 7.25-7.15 (m, 10H), 6.26-6.23 (m, 1H), 5.87-5.73 (m,4H), 2.27 (s, 3H), 1.69 (d, 1H, J=6.8 Hz), 1.46 (d, 1H, J=6.4 Hz).

Step 6:(1R,3aS,6S,7R,7aS)-7-(diphenylcarbamoyl)-1,6-dimethyl-3-oxo-1,3,3a,6,7,7a-hexahydroisobenzofuran-5-ylacetate: (2E,4Z)-(R,Z)-5-(Diphenylamino)-5-oxopent-3-en-2-yl4-acetoxyhexa-2,4-dienoate (100 g, 0.24 mol) in NMP (2.5 L) was stirredat 145° C. for 2 h. The TLC (petroleum ether:ethyl acetate, 3:1) showedthe reaction was almost complete. The reaction was cooled to 50° C. andDBU (3.6 mL, 2.39 mmol) was added in one portion. After 1 h, thereaction was cooled to 20° C. and was poured to cold water (22 L). Thereaction was extracted with ethyl acetate (22 L). The organic layer waswashed with water (22 L×2). The combined aqueous layers were extractedwith ethyl acetate (5 L×3). All the organic layers from the tenreactions run were then combined together, washed with brine and driedwith anhydrous Na₂SO₄. After concentration, a precipitate that hadformed was washed with methyl tert-butyl ether to give the titlecompound. The filtrate was concentrated and purified by silica gelcolumn chromatography (petroleum ether:CH₂Cl₂, 6:1) to give anotherbatch of the title compound. ¹H NMR (400 MHz, CDCl₃) δ 7.51-7.23 (m,10H), 5.26 (s, 1H), 4.90-4.86 (m, 1H), 3.21-3.10 (m, 2H), 2.83 (dd, 1H,J=10.8, 3.6 Hz), 2.52-2.50 (m, 1H), 2.16 (s, 3H), 1.58 (d, 3H, J=6.0Hz), 1.09 (d, 3H, J=6.4 Hz).

Step 7:(3R,3aS,4R,5S,7aR)-3,5-dimethyl-1,6-dioxo-N,N-diphenyloctahydroisobenzofuran-4-carboxamide: HCl (298 mL, 4 M in water) was added dropwise to(1R,3aS,6S,7R,7aS)-7-(diphenylcarbamoyl)-1,6-dimethyl-3-oxo-1,3,3a,6,7,7a-hexahydroisobenzofuran-5-ylacetate (100 g, 238 mmol) in CH₃OH (1 L) at 0° C. After the addition,the reaction was warmed to room temperature and stirred for another 36h. The TLC (petroleum ether:ethyl acetate, 3:1) showed the reaction wasalmost complete. Methanol was removed under reduced pressure at 35° C.The reaction was extracted with CH₂Cl₂ and the organics was washed withsat. NaHCO₃, brine, dried over Na₂SO₄ and concentrated. The resultantresidue was purified on multiple 15 g scale silica gel columns(petroleum ether:CH₂Cl₂, 6:1) to yield the title compound. MS ESI calcd.for C₂₃H₂₄NO₄ [M+H]⁺ 378, found 378. ¹H NMR (400 MHz, CDCl₃) δ 7.50-7.46(m, 3H), 7.35-7.33 (m, 2H), 7.29-7.22 (m, 5H), 5.16-5.10 (m, 1H),2.98-2.90 (m, 3H), 2.65-2.63 (m, 1H), 2.55-2.46 (m, 2H), 1.56 (d, 3H,J=5.6 Hz), 1.18-1.14 (m, 3H).

Step 8:(3R,3aS,4R,5S,7aR)-6,6-difluoro-3,5-dimethyl-1-oxo-N,N-diphenyloctahydroisobenzofuran-4-carboxamide:To a solution of(3R,3aS,4R,5S,7aR)-3,5-dimethyl-1,6-dioxo-N,N-diphenyloctahydroisobenzofuran-4-carboxamide(300.0 g, 0.79 mol) in CH₂Cl₂ (anhyd., 3 L) was added DAST (180 mL, 2.37mol) dropwise slowly at 15-30° C. The resulting mixture was stirredovernight at 25° C. After LCMS showed the mixture was complete, themixture was slowly poured to a solution of K₃PO₄.3H₂O (0.4 mol/L, 3 L)and was partitioned with water and CH₂Cl₂. The aqueous layer wasextracted with CH₂Cl₂ (1000 mL×2). The combined organic layers werewashed with NaHCO₃ (500 mL), brine (500 mL), dried over Na₂SO₄, filteredand concentrated under reduced pressure. The residue was oxidized byKMnO₄ (100 g) in DMAC for 2 hours, then filtered. The filtrate wasextracted by EtOAc, washed with 10% CaCl₂ aqueous solution, and brine.The combined organic solution was dried by Na₂SO₄ and concentrated. Theresidue was further purified by recrystallization with ethanol (3 V) togive the title compound. ¹H NMR (400 MHz, CDCl₃) δ 7.40 (m, 3H), 7.24(m, 7H), 4.84 (m, 1H), 2.76 (m, 1H), 2.45 (m, 3H), 2.18 (m, 1H), 1.71(m, 1H), 1.5 (d, 3H, J=5.6 Hz), 1.1 (d, 3H, J=6.5 Hz).

Step 9:(3R,3aR,4R,5S,7aR)-6,6-difluoro-3,5-dimethyl-1-oxooctahydroisobenzofuran-4-carboxylicacid: To a solution of the(3R,3aS,4R,5S,7aR)-6,6-difluoro-3,5-dimethyl-1-oxo-N,N-diphenyloctahydroisobenzofuran-4-carboxamide(260 g, 0.653 mol) in THF (1300 mL) was added a solution of LiOH.H₂O (55g, 1.31 mol) in H₂O (650 mL) at room temperature. The mixture was heatedto 60° C. and stirred for 2 h. Upon completion of reaction, the mixturewas diluted with LiOH.H₂O solution (1.3 L, 10% in water). The THF layerwas removed in vacuo. The aqueous phase was extracted with MTBE (800mL×3). The aqueous layer was acidified to pH 1-2 with 1N HCl andextracted with EtOAc (800 mL mL×3). The combined organic layers werewashed with water (500 mL), brine (500 mL), dried over Na₂SO₄, filteredand concentrated to give the title compound. ¹H NMR (400 MHz, CDCl₃) δ4.73 (m, 1H), 2.99 (m, 1H), 2.89 (m, 1H), 2.64 (m, 1H), 2.53 (m, 1H),2.33 (m, 1H), 1.86 (m, 1H), 1.39 (d, 3H), 1.15 (d, 3H).

Step 10:(3R,3aS,4R,5S,7aR)-6,6-difluoro-4-(hydroxymethyl)-3,5-dimethylhexahydroisobenzofuran-1(3H)-one:To a solution of(3R,3aR,4R,5S,7aR)-6,6-difluoro-3,5-dimethyl-1-oxooctahydroisobenzofuran-4-carboxylicacid (81.0 g, 0.326 mol) in CH₂Cl₂ (800 mL) was added (COCl)₂ (58.8 mL,0.66 mol) and DMF (1 mL) at 20-25° C. under nitrogen. The mixture wasstirred for 4 hours. Upon reaction completion, the mixture wasconcentrated under reduced pressure. The residue was dissolved with THF(400 mL×2) and then concentrated twice. The residue was dissolved in THF(500 mL), and a solution of LiAlH(t-BuO)₃ (653 mL, 0.653 mol, 1 M inTHF) was added dropwise slowly below −55° C. under nitrogen. The mixturewas slowly warmed to room temperature and stirred overnight. Uponreaction completion, the mixture was quenched with 1N HCl (1 L) andextracted with EtOAc (500 mL×3). The combined organic layers were washedwith water (500 mL), brine (500 mL), dried over Na₂SO₄, filtered andconcentrated to give the title compound. ¹H NMR (400 MHz, CDCl₃) δ 4.77(m, 1H), 3.83 (m, 2H), 2.85 (m, 1H), 2.45 (m, 2H), 2.07 (m, 2H), 1.83(m, 1H), 1.59 (d, 3H), 1.13 (d, 3H).

Step 11:(3R,3aS,4R,5S,7aR)-6,6-difluoro-3,5-dimethyl-1-oxooctahydroisobenzofuran-4-carbaldehyde:To a stirred solution of(3R,3aS,4R,5S,7aR)-6,6-difluoro-4-(hydroxymethyl)-3,5-dimethylhexahydroisobenzofuran-1(3H)-one(56 g, 0.24 mol) in MeCN (600 mL) was added Dess-Martin reagent (122 g,0.287 mol) and NaHCO₃ (60.3 g, 227 mol) under nitrogen at 0° C. Themixture was stirred for 4 h at 25° C. Upon reaction completion, themixture was transferred to into L-ascorbic acid (5% aq., 1500 mL) undernitrogen, and then was filtered. The filtrate was quenched with Na₂SO₃(5% aq., 750 mL). The solvent was removed and the product was extractedwith EtOAc (750 mL×3). The combined organic layers were washed withbrine (900 mL), dried over Na₂SO₄, filtered and concentrated to give thetitle compound, which was directly used in the next step without furtherpurification.

(3R,3aS,4R,5S,7aR)-4-((E)-2-(5-bromopyridin-2-yl)vinyl)-6,6-difluoro-3,5-dimethylhexahydroisobenzofuran-1(3H)-one:To a solution of diethyl ((5-bromopyridin-2-yl)methyl)phosphonate (95.6g, 0.310 mol) in THF (400 mL) was added LiHMDS (310 mL, 0.310 mol, 1 Min THF) dropwise at 0° C. under nitrogen. The mixture was stirred for 30min at 0° C., and then warmed up to about 25° C. Ti(OiPr)₄ (110 g,0.3103 mmol) was added and the reaction was stirred for 30 min at 25° C.A solution of(3R,3aS,4R,5S,7aR)-6,6-difluoro-3,5-dimethyl-1-oxooctahydroisobenzofuran-4-carbaldehyde(36 g, 0.1552 mmol) in THF (400 mL) was added into the mixture, andstirred overnight at room temperature. Upon reaction completion, themixture was quenched with saturated solution of potassium sodiumtartrate (1 L), and then filtered. The filtrate was extracted with EtOAc(500 mL×3). The combined organic layers were washed with brine (500 mL),dried over Na₂SO₄, filtered and concentrated. The residue was purifiedby column chromatography with PE/EtOAc (20:1) to give the titlecompound. MS ESI calcd. for C₁₇H₁₉BrF₂NO₂ [M+H]⁺ 386/388, found 386/388.¹H NMR (500 MHz, CDCl₃) δ 8.61 (d, 1H, J=2 Hz), 7.78 (dd, 1H, J=2.4, 8.4Hz), 7.09 (d, 1H, J=8.4 Hz), 6.55 (m, 2H), 4.74 (m, 1H), 2.95 (m, 1H),2.73 (m, 1H), 2.38-2.53 (m, 2H), 1.83-2.04 (m, 2H), 1.45 (d, 3H, J=6Hz), 1.07 (d, 3H, J=6.8 Hz).

Intermediate B can be prepared according to Scheme B through anoxidation of Intermediate A via an anionic trapping of oxygen.

Intermediate B

(3R,3aR,4R,5S,7aS)-4-((E)-2-(5-bromopyridin-2-yl)vinyl)-6,6-difluoro-7a-hydroxy-3,5-dimethylhexahydroisobenzofuran-1(3H)-one:Lithium bis(trimethylsilyl)amide (25.2 mL, 25.2 mmol) was added to adegassed THF (100 mL) solution of intermediate B1 (7.5 g, 19.42 mmol) at0° C. The reaction was stirred for 25 min before the system was purgedand flushed 3× with oxygen. The reaction was kept under oxygen and wasstirred for 0.5 hour at 0° C. and then bath was removed and reactionallowed to warm to 25° C. After 1 hour, the reaction was partitionedbetween EtOAc (200 mL) and 10% aqueous NaHSO₃ (200 mL). The organic waswashed with sat'd aqueous NaHCO₃ (200 mL), brine (200 mL), and driedover sodium sulfate and concentrated prior to silica gel chromatography(0-100% acetone in hexanes) to obtain the title compound. MS ESI calcd.for C₁₇H₁₉BrF₂NO₃ [M+H]⁺ 402/404, found 402/404. ¹H NMR (500 MHz, CDCl₃)δ 8.65 (d, J=2.2 Hz, 1H), 7.82 (dd, J=8.3, 2.4 Hz, 1H), 7.12 (d, J=8.3Hz, 1H), 6.66-6.58 (m, 2H), 4.67-4.61 (m, 1H), 3.51 (s(br), 1H),2.97-2.92 (m, 1H), 2.49-2.41 (m, 2H), 2.14-2.06 (m, 2H), 1.51 (d, J=6.0Hz, 3H), 1.13 (d, J=6.6 Hz, 3H).

Intermediate C can prepared according to Scheme C from through apalladium-mediated boronylation of Intermediate B.

Intermediate C

3R,3aR,4R,5S,7aS)-6,6-difluoro-7a-hydroxy-3,5-dimethyl-4-((E)-2-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)vinyl)hexahydroisobenzofuran-1(3H)-one:In the reaction vessel(3R,3aR,4R,5S,7aS)-4-((E)-2-(5-bromopyridin-2-yl)vinyl)-6,6-difluoro-7a-hydroxy-3,5-dimethylhexahydroisobenzofuran-1(3H)-one(5.0 g, 12.4 mmol) was combined with potassium acetate (2.44 g, 24.9mmol) and bispinacolatodiboron (4.42 g, 17.4 mmol). This mixture wasthen evacuated and backfilled with N₂ (3 times). Then dry, degasseddioxane (62.2 mL) was added to this flask. This mixture was then heatedat 90° C. for 0.5 h before cooling to 25° C. and diluting with 60 mLacetonitrile. The reaction was filtered and was concentrated to yield(3R,3aR,4R,5S,7aS)-6,6-difluoro-7a-hydroxy-3,5-dimethyl-4-((E)-2-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyridin-2-yl)vinyl)hexahydroisobenzofuran-1(3H)-onethat was used without further purification. MS ESI calcd. forC₁₇H₁₉BF₂NO₃ [M+H]⁺ (ionizes for boronic acid) 368, found 368. ¹H NMR(499 MHz, CDCl₃) δ 8.92 (d; J=5.30 Hz; 1H); 8.06 (t; J=6.63 Hz; 1H);7.30 (d; J=5.86 Hz; 1H); 7.21 (t; J=6.85 Hz; 1H); 6.66-6.71 (m; 2H);4.61-4.65 (m; 1H); 3.73-3.74 (m; 16H); 2.95 (dd; J=12.13; 6.89 Hz; 1H);2.43 (br s; 3H); 2.10-2.12 (m; 2H); 2.04 (t; J=6.05 Hz; 1H); 1.38 (d;J=5.58 Hz; 13H); 1.27-1.31 (m; 46H); 1.14 (t; J=6.20 Hz; 3H).

General Synthetic Schemes

Representative compounds of the present invention can be synthesizedaccording to the general schemes outlined below as well as therepresentative examples that follow. Since the schemes are anillustration, the invention should not be construed as being limited bythe chemical reactions and conditions expressed. The preparation of thevarious starting materials used in the schemes is well within theordinary skill level of a practitioner of this art. Unless otherwiseindicated, the definition for a variable is the same as that provided inFormula I.

Compounds of Formula (1) can be prepared according to Scheme 1 fromIntermediate C by a palladium-mediated coupling with known orcommercially available bromides.

Compounds of Formula (2) can be prepared according to Scheme 2 fromIntermediates A or B by a palladium-mediated coupling with known orcommercially available boronic acids or esters.

Compounds of Formula (3) can be prepared according to Scheme 3 fromcompound 3-1 (Example 4) by a palladium-mediated Negishi-type couplingreaction.

Compounds of Formula (4) can be prepared according to Scheme 4 fromcompound 3-1 (Example 4) by a palladium-mediated coupling reaction withcommercially available amines.

EXAMPLES

The following schemes and examples are provided so that the inventionwill be more fully appreciated and understood. Starting materials aremade using known procedures or as illustrated below.

Example 1

6′-((E)-2-((3R,3aR,4R,5S,7aS)-6,6-difluoro-7a-hydroxy-3,5-dimethyl-1-oxooctahydroisobenzofuran-4-yl)vinyl)-[3,3′-bipyridine]-2-carbonitrile: To anitrogen purged mixture of intermediate C (5.6 g, 12.46 mmol),3-bromopicolinonitrile (4.56 g, 24.93 mmol) and K₂CO₃ (5.17 g, 37.4mmol) in dioxane (50 mL)/water (10 mL) was added1,1′-bis(di-tert-butylphosphino)ferrocene palladium dichloride (0.812 g,1.25 mmol) under nitrogen at 25° C. The reaction mixture was placed in50° C. bath and was stirred for 0.5 hour. The reaction mixture wascooled to 25° C., partitioned between EtOAc (400 mL) and water (400 mL).The organic layer was washed with brine (400 mL), dried over sodiumsulfate and concentrated prior to purification by silica gelchromatography (0-100% acetone in hexanes) to yield the title compound.MS ESI calcd. for C₂₃H₂₂F₂N₃O₃ [M+H]⁺ 426, found 426. ¹H NMR (500 MHz,CDCl₃) δ 8.81-8.79 (m, 2H), 8.00 (dd, J=8.1, 2.3 Hz, 1H), 7.94 (d,J=8.03 Hz, 1H), 7.69 (dd, J=8.01, 4.73 Hz, 1H), 7.41 (d, J=8.11 Hz, 1H),6.79-6.72 (m, 2H), 4.67-4.65 (m, 1H), 3.45 (s, 1H), 3.04-2.99 (m, 1H),2.49-2.38 (m, 2H), 2.13-2.09 (m, 2H), 1.56 (d, J=6.0 Hz, 3H), 1.18 (d,J=6.59 Hz, 3H). PAR-1 FLIPR IC₅₀=5.9 nM.

The following examples in Table 1 were prepared according to Scheme 1using the procedure outlined in the synthesis of Example 1 using knownor commercially available bromides.

TABLE 1 PAR-1 Exact Mass FLIPR Ex Structure IUPAC Name [M + H]+ IC₅₀(nM) 2

(3R,3aR,4R,5S,7aS)-6,6- difluoro-7a-hydroxy-3,5- dimethyl-4-((E)-2-(2′-phenyl-[3,3′-bipyridin]-6- yl)vinyl)hexahydroiso- benzofuran-1(3H)-one477 9.0

Example 3

(3R,3aR,4R,5S,7aS)-6,6-difluoro-7a-hydroxy-4-((E)-2-(2′-methoxy-[3,3′-bipyridin]-6-yl)vinyl)-3,5-dimethylhexahydroisobenzofuran-1(3H)-one:To a mixture of (2-methoxypyridin-3-yl)boronic acid (19.0 mg, 0.124mmol), Intermediate B (50 mg, 0.124 mmol) and K₂CO₃ (51.5 mg, 0.373mmol) in dioxane (0.9 mL)/water (0.1 mL) was added1,1′-bis(di-tert-butylphosphino)ferrocene palladium chloride (8.10 mg,0.012 mmol) at RT. The reaction mixture was flushed and purged with N₂(3×) and was heated to 60° C. for 1 hour. The reaction mixture wascooled to 25° C., diluted with acetonitrile (3 mL) and was filteredbefore concentrating and purification by HPLC (10-100%acetonitrile/water with 0.1% NH₄OH modifier) to provide the titlecompound. MS ESI calcd. for C₂₃H₂₅F₂N₂O₄ [M+H] 431, found 431. ¹H NMR(500 MHz, CDCl₃): δ 8.79 (d; J=2.22 Hz; 1H); 8.24 (dd; J=4.98; 1.86 Hz;1H); 7.92 (dd; J=8.07; 2.30 Hz; 1H); 7.67 (dd; J=7.32; 1.89 Hz; 1H);7.05 (dd; J=7.32; 4.99 Hz; 1H); 6.71-6.61 (m; 2H); 4.68-4.62 (m; 1H);4.02 (s; 3H); 3.25 (s; 1H); 2.48-2.43 (m; 2H); 2.16-2.05 (m; 2H); 1.55(d; J=6.00 Hz; 3H); 1.16 (d; J=6.59 Hz; 3H). PAR-1 FLIPR IC₅₀=5.4 nM.

The following examples in Table 2 were prepared according to Scheme 2using the procedure outlined in the synthesis of Example 3 using knownor commerically available boronic acids or esters.

TABLE 2 PAR-1 Exact Mass FLIPR Ex Structure IUPAC Name [M + H]+ IC₅₀(nM) 4

(3R,3aR,4R,5S,7aS)-4- ((E-2-(2′-chloro-[3,3′- bipyridin]-6-yl)vinyl)-6,6-difluoro-7a- hydroxy-3,5- dimethylhexahydroiso benzofuran-1(3H)-one435  7.8 5

(3R,3aR,4R,5S,7aS)-4- ((E)-2-(2′-fluoro-[3,3′- bipyridin]-6-yl)vinyl)-7a-hydroxy-3,5- dimethylhexahydroiso benzofuran-1(3H)-one 419 16.2

Example 6

(3R,3aR,4R,5S,7aS)-4-((E)-2-(2′-cyclopropyl-[3,3′-bipyridin]-6-yl)vinyl)-6,6-difluoro-7a-hydroxy-3,5-dimethylhexahydroisobenzofuran-1(3H)-one:To a 2 mL microwave vial was added(3R,3aR,4R,5S,7aS)-4-((E)-2-(2′-chloro-[3,3′-bipyridin]-6-yl)vinyl)-6,6-difluoro-7a-hydroxy-3,5-dimethylhexahydroisobenzofuran-1(3H)-one(30 mg, 0.069 mmol),(2-dicyclohexylphosphino-2′,4′,6′-triisopropyl-1,1′-biphenyl)[2-(2-aminoethyl)phenyl]palladium(II)chloride (5.10 mg, 6.90 μmol), cyclopropylzinc(II) bromide (15 mg, 0.08mmol) and tetrahydrofuran (1 mL). The resulting reaction mixture wasirradiated in a Biotage® Initiator for 45 min at 150° C. The reactionmixture was washed with water (2 mL) and was extracted with EtOAc (2×3mL). The combined organic layers were concentrated to give a residue,which was dissolved in DCM (2 mL) and 50 mg of SiliaMetS®Dimercaptotriazine (DTM) (SiliCycle, Inc. CAT#: R79030B) was added andshaken for 12 hours at room temperature to remove palladium catalyst.The mixture was filtered and the filtrate was concentrated to give aresidue which was dissolved in 2 mL of DMSO. The solution was filteredand purified by mass triggered reverse phase HPLC (ACN/water with 0.1%TFA modifier) to afford the title compound. MS ESI calcd. forC₂₅H₂₇F₂N₂O₃ [M+H]⁺ 441, found 441. ¹H NMR (500 MHz, DMSO) δ 8.68 (s,1H), 8.43 (d, 1H, J=7.5 Hz), 7.89 (dd, 1H, J=1.5, 7.5 Hz), 7.65 (d, 1H,J=7.5 Hz), 7.58 (d, 1H, J=7.5 Hz), 7.22 (dd, 1H, J=1.5, 7.5 Hz), 6.76(dd, 1H, J=6.1, 15.1 Hz), 6.06 (d, 1H, J=15.1 Hz), 4.85 (m, 1H), 2.78(m, 1H), 2.60-2.20 (m, 6H), 2.06 (m, 1H), 1.92 (m, 1H), 1.38 (d, 3H,J=6.8 Hz), 1.06 (d, 1H, J=7.0 Hz), 0.99 (d, 1H, J=6.0 Hz). 0.96 (d, 3H,J=6.8 Hz). PAR-1 FLIPR IC₅₀=10.4 nM.

The following examples in Table 3 were prepared according to Scheme 3using the procedure outlined in the synthesis of Example 6 using knownor commerically available organozinc compounds.

TABLE 3 PAR-1 Exact Mass FLIPR Ex Structure IUPAC Name [M + H]+ IC₅₀(nM) 7

(3R,3aR,4R,5S,7aS)- 6,6-difluoro-7a- hydroxy-3,5-dimethyl-4-((E)-2-(2′-(5- methylthiophen-2-yl)- [3,3′-bipyridin]-6-yl)vinyl)hexahydroiso benzofuran-1(3H)-one 497 15.17 7

(3R,3aR,4R,5S,7aS)- 6,6-difIuoro-7a- hydroxy-3,5-dimethyl-4-((E)-2-(2′-(l- methyl-1H-imidazol- 5-yl)-[3,3′-bipyridin]-6-yl)vinyl)hexahydro isobenzofuran-1(3H)- one 481 200   

Example 9

(3R,3aR,4R,5S,7aS)-4-((E)-2-(2′-(3,3-difluoroazetidin-1-yl)-[3,3′-bipyridin]-6-yl)vinyl)-6,6-difluoro-7a-hydroxy-3,5-dimethylhexahydroisobenzofuran-1(3H)-one:To a 1 dram vial was charged (3R,3aR,4R,5S,7aS)-4-((E)-2-(2′-chloro-[3,3′-bipyridin]-6-yl)vinyl)-6,6-difluoro-7a-hydroxy-3,5-dimethylhexahydroisobenzofuran-1(3H)-one(50 mg, 0.115 mmol), 3,3-difluoroazetidine.HCl (29.8 mg, 0.230 mmol),Ruphos Indoline Precatalyst (16.76 mg, 0.023 mmol), and Cs₂CO₃ (150 mg,0.460 mmol) in tert-amyl alochol (1.5 mL). The resulting reactionmixture was stirred for 16 hours (overnight) at 80° C. Then, thereaction was filtered, diluted with EtOAc (6 mL) and the resultingsolution washed by saturated aq. Na₂CO₃(2×4 mL) and water (2×4 mL). Theorganic layer was dried over anhydrous Na₂SO₄, concentrated in vacuo togive a residue, which was dissolved in 1 mL of DCM and purified by flashchromatography on silica gel (EtOAc/hexanes) to yield the titlecompound. MS ESI calcd. for C₂₅H₂₆F₄N₃O₃ [M+H]⁺ 492, found 492. PAR-1FLIPR IC₅₀=92.4 nM

Assays

The following assays were used to evaluate the ability of the inventivecompounds to act as PAR-1 receptor antagonists, their interactions withother therapeutic agents in the body or their inhability to causedrug-induced long QT syndrome.

PAR-1 FLIPR Assay

This assay measures the potency of the inventive compounds as PAR-1receptor antagonists.

Frozen HEK 293 Cells were plated in 384-well PDL coated plates at 12000cells/well in 50 uL of DMEM media containing 10% FBS,pen/strep/L-Glutamine and non-essential amino acids, incubated overnightat 37° C./5% CO₂. Media was then removed from the cells, incubated with33 uL of Calcium-5 dye in assay buffer (Hank's buffer containing 20 mMHEPES, 0.04% Chaps and 2.5 mM Probenecid) for 60 minutes at 37° C. 2 uLof varying concentrations of compound in 40% DMSO in assay buffer (finalDMSO concentration is 2.3%) were then added to the cells and incubatedat 25° C. for 30 minutes. The plates were added to the FLIPR Tetra®, thedevice added 5 uL of PAR-1 selective receptor-activating peptide(sequence Ala-parafluoroPhe-Arg-Cha-Cit-Try-Nh2, prepared in water) at aconcentration equal to the effective concentration that achieved 80%activation of signaling on the day of the experiment. The range ofpeptide was from 1.5-3 μM. The final volume was 40 uL/well, with 2%DMSO. The FLIPR was read at an excitation wavelength of 480 nm and anemission wavelength of 535 nm, and performed 60 scans over a 1-2 minreading time. The data were analyzed by taking the peak signal over aportion of the range of the 60 scans and dividing this signal by theminimum signal for that same range. The data were expressed as percentinhibition of the maximum divided by the minimum signal achieved at 80%activation produced by the PAR1 activating peptide on the test day. Thecompounds of Examples 1-9 were tested in the assay described above andthe data collected for these compounds is provided.

CYP MUX (3A4) RI

This assay measures inhibition of CYP3A4 by a compound. Cytochromes P450(CYPs) constitute a superfamily of heme-containing enzymes thatrecognize and metabolize a large number structurally diverse xenobioticsin the human body. CYP3A4 constitutes the largest portion of CYP enzymesin the liver that accounts for the metabolism of almost 50% of alldrugs. This assay is used to evaluate the potential of a compound fordeveloping DDIs. (Clarke, S. E.; Jones B. C. Drug-Drug Interactions, NewYork: Marcel Dekker; 2002. pp. 53-88).

Compound dilutions and assay-ready plates were prepared on a TTP LabtechMosquito® HTS. Assay conduction was fully automated on a customizedScreening Platform from Caliper (now PerkinElmer) containing aMitsubishi robotic plate handler, Liconic incubators, a Caliper Zephyr®liquid handling workstation equipped with temperature-controlled deckpositions, a Biotek MultiFlo™ dispenser and an Agilent PlateLoc heatsealer. Assay plates were Corning Costar® 384 well PP plates. Highthroughput mass spectrometric readout was performed on a RapidFire® 300system coupled to an AB Sciex API 4000™ triple quadrupole device. CYPisoform 3A4 was incubated in a separate reaction of 50 μL final volume.25 μL of HLM (human liver microsomes, BD UltraPool™ 150, 0.25 mg/mLfinal concentration) and the respective substrate, testosterone (75 μM)for 3A4, in potassium phosphate buffer (100 mM, pH=7.4) were added to250 nL of stamped compound solution (10 mM in DMSO). The reactions werestarted upon addition of 25 μL of a co-factor solution containingmagnesium chloride (3.3 mM), glucose-6-phosphate (3.3 mM),glucose-6-phosphate dehydrogenase (1.4 units) and NADP (1 mM) inpotassium phosphate buffer (100 mM, pH=7.4) and incubated on deck at 37°C. for 10 min. 8 μL of each reaction were transferred to the samereadout plated filled with 48 μL of stop solution containing internalstandards (concentration in final readout plate),6-hydroxytestosterone-D7 (0.5 μM), 4′-hydroxydiclofenac-D4′-(0.2 μM),and dextrorphan-D3 (0.01 μM), in acetonitrile with 0.5% formic acid.After heat sealing, plates were stored at −20° C. for at least 30 min,centrifuged and subjected directly to RapidFire®/MS analysis.

MK-499 Filter Binding Assay

This assay is used to evaluate the potential of a compound to causedrug-induced log QT syndrome.

Drug cardiac arrhythmia is an important safety concern forpharmaceutical development and health regulatory authorities. Blockadeof heterologously-expressed human ether-à-go-go gene (hERG) channelprolongs the duration of the cardiac action potential leading to a longQT interval that can lead to sudden death (De Ponti, F.; et at DrugSafety 2002, 25, pp. 263-286). It is important to have compounds devoidof hERG channel activity as measured by an in vitro assay. Affinity ofcompounds for the hERG channel was evaluated in radioligand competitionexperiments using HEK293 cells that were stably transfected with thehERG channel and radiolabeled ligand, MK-499 a potent antiarrhythmic.This assay correlates well with QT prolongation in vivo (Jamieson, C.;et al., J. Med. Chem. 2006, 49, pp. 5029-5046).

25 μL Target membranes (in assay buffer: 10 mM HEPES/NaOH, pH 7.4, 70 mMNaCl, 60 mM KCl, 2 mM MgCl₂, 1 mM CaCl₂) purified from a HEK293 cellline expressing the human Ether-à-go-go Related Gene (hERG) ion channel,1 μL test compound in 10 mM DMSO and 25 μL (6,000 cpm/well; in assaybuffer) [35S]MK-0499 radioligand (Merck/Perkin Elmer) are added to theassay plate (Axygen; 384 Deep well “Diamond Plate”, clear). Afterincubation of the binding reaction at room temperature (RT) for 90 min50 μL of the assay were transferred to a MultiscreenHTS 384 FC filterplate (Millipore), which has been pre-wetted with 20 μL 0.01% PEI/0.01%Triton X-100 for at least 30 min at RT. Then, 30 μL it wash buffer (10mM HEPES/NaOH, pH 7.4, 130 mM NaCl, 2 mM MgCl₂, 1 mM CaCl₂) equilibratedto RT were added to each well of the assay plate and subsequentlytransferred to the filter plate. The assay mixture was aspirated throughthe filter plate using a Biotek ELx405™ washer. The filter plate waswashed twice with 100 μl cold wash buffer per wash and well and thendried in a drying oven for at least 75 min at 55° C. Afterwards, thebottom of each filter plate was heat sealed with a solid foil seal, then10 μL of Microscint™ 0 (Perkin Elmer) were added to each well of thefilter plate and finally, the top of each filter plate was sealed with aclear seal. The plates were stored for at least 20 min in a MicroBeta2reader (Perkin Elmer) before they are counted (60 sec/well).

Table 4 summarizes the selectivity of the inventive compounds in theCYP3A4 assay and the ability of the inventive compound to inhibit hERG.

TABLE 4 Ex CYP2D6 IC₅₀ (μM) MK499 IC₅₀ (μM) 1 50 60 2 28.5 39.8 3 50 604 50 18.7 5 50 60 6 50 60 7 50 20.8 8 50 60 9 50 60

While the invention has been described with reference to certainparticular embodiments thereof, numerous alternative embodiments will beapparent to those skilled in the art from the teachings describedherein. Recitation or depiction of a specific compound in the claims(i.e., a species) without a specific stereoconfiguration designation, orwith such a designation for less than all chiral centers, is intended toencompass the racemate, racemic mixtures, each individual enantiomer, adiastereoisomeric mixture and each individual diastereomer of thecompound where such forms are possible due to the presence of one ormore asymmetric centers. All patents, patent applications andpublications cited herein are incorporated by reference in theirentirety.

We claim:
 1. A compound of the formula

or a pharmaceutically acceptable salt thereof wherein: R¹ is halo; —CN;alkyl; cycloalkyl; alkoxy; phenyl, which is optionally substituted oneor twice independently by alkyl, halo, or —CN; or a thiophene ring,which is optionally substituted once or twice independently by alkyl. 2.The compound as defined in claim 1 or a pharmaceutically acceptable saltthereof wherein R¹ is methoxy, —CN, —F, —Cl, or cyclopropyl.
 3. Acompound as defined in claim 1, which is:6′-((E)-2-((3R,3aR,4R,5S,7aS)-6,6-difluoro-7a-hydroxy-3,5-dimethyl-1-oxooctahydroisobenzofuran-4-yl)vinyl)-[3,3′-bipyridine]-2-carbonitrile;(3R,3aR,4R,5S,7aS)-6,6-difluoro-7a-hydroxy-3,5-dimethyl-4-((E)-2-(2′-phenyl-[3,3′-bipyridin]-6-yl)vinyl)hexahydroisobenzofuran-1(3H)-one;(3R,3aR,4R,5S,7aS)-6,6-difluoro-7a-hydroxy-4-((E)-2-(2′-methoxy-[3,3′-bipyridin]-6-yl)vinyl)-3,5-dimethylhexahydroisobenzofuran-1(3H)-one;(3R,3aR,4R,5S,7aS)-4-((E)-2-(2′-chloro-[3,3′-bipyridin]-6-yl)vinyl)-6,6-difluoro-7a-hydroxy-3,5-dimethylhexahydroisobenzofuran-1(3H)-one;(3R,3aR,4R,5S,7aS)-4-((E)-2-(2′-fluoro-[3,3′-bipyridin]-6-yl)vinyl)-7a-hydroxy-3,5-dimethylhexahydroisobenzofuran-1(3H)-one;(3R,3aR,4R,5S,7aS)-4-((E)-2-(2′-cyclopropyl-[3,3′-bipyridin]-6-yl)vinyl)-6,6-difluoro-7a-hydroxy-3,5-dimethylhexahydroisobenzofuran-1(3H)-one;3R,3aR,4R,5S,7aS)-6,6-difluoro-7a-hydroxy-3,5-dimethyl-4-((E)-2-(2′-(5-methylthiophen-2-yl)-[3,3′-bipyridin]-6-yl)vinyl)hexahydroisobenzofuran-1(3H)-one;or a pharmaceutically acceptable salt thereof.
 4. A pharmaceuticalcomposition comprising an effective amount of a compound as defined inclaim 1 or a pharmaceutically acceptable salt thereof and apharmaceutically acceptable carrier.
 5. The pharmaceutical compositionas defined in claim 4, which further comprises a therapeuticallyeffective amount of at least one additional cardiovascular agentselected from the group consisting of: aspirin, seratrodast, picotamide,ramatroban, clopidogrel, meloxicam, rofecoxib, celecoxib, valsartan,telmisartan, candesartran, irbesartran, losartan, eprosartan,tezosentan, milrinoone, enoximone, captopril, enalapril, enaliprilat,spirapril, quinapril, perindopril, ramipril, fosinopril, trandolapril,lisinopril, moexipril, benazepril, candoxatril, ecadotril, ximelagatran,fondaparin, enoxaparin, chlorothiazide, hydrochlorothiazide, ethacrynicacid, furosemide, amiloride, abciximab and eptifibatide.
 6. Thepharmaceutical composition as defined in claim 5, wherein the at leastone additional cardiovascular agent is aspirin or clopidogrel, whereinclopidogrel is a free base or pharmaceutically acceptable salt.
 7. Apharmaceutical composition comprising an effective amount of a compoundas defined in claim 3 or a pharmaceutically acceptable salt thereof anda pharmaceutically acceptable carrier.
 8. The pharmaceutical compositionas defined in claim 7, which further comprises a therapeuticallyeffective amount of at least one additional cardiovascular agentselected from the group consisting of: aspirin, seratrodast, picotamide,ramatroban, clopidogrel, meloxicam, rofecoxib, celecoxib, valsartan,telmisartan, candesartran, irbesartran, losartan, eprosartan,tezosentan, milrinoone, enoximone, captopril, enalapril, enaliprilat,spirapril, quinapril, perindopril, ramipril, fosinopril, trandolapril,lisinopril, moexipril, benazepril, candoxatril, ecadotril, ximelagatran,fondaparin, enoxaparin, chlorothiazide, hydrochlorothiazide, ethacrynicacid, furosemide, amiloride, abciximab and eptifibatide.
 9. Thepharmaceutical composition as defined in claim 8, wherein the at leastone additional cardiovascular agent is aspirin or clopidogrel, whereinclopidogrel is a free base or pharmaceutically acceptable salt.
 10. Amethod for treating acute coronary syndrome or peripheral artery diseaseby administering a compound as defined in claim 1 to a mammal in need ofsuch treatment.
 11. A method of inhibiting platelet aggregationcomprising administering to a mammal an effective amount of a compoundas defined in claim
 1. 12. A method for treating acute coronary syndromeor peripheral artery disease by administering a compound as defined inclaim 3 or a pharmaceutically acceptable salt thereof to a mammal inneed of such treatment.
 13. A method of inhibiting platelet aggregationcomprising administering to a mammal an effective amount of compound asdefined in claim 3 or a pharmaceutically acceptable salt thereof.